Derivatization of pBACpAK entrapment vectors for enhanced mobile genetic element transposition detection in multidrug-resistant Escherichia coli

<b>Aim.</b> Antimicrobial resistance poses a critical global health threat, driven by the dissemination of resistance genes via mobile genetic elements (MGEs). This study aims to enhance the detection of MGE insertions in multidrug-resistant <i>Escherichia coli</i> by derivatizing the pBACpAK entrapment vector. <b>Methods and results.</b> Three derivatives were constructed with additional nucleotides upstream of the <i>cI</i> repressor gene, based on conserved regions identified from GenBank sequences containing known IS<i>26</i> and IS<i>1</i> insertions. Using colony PCR, intracellular transposition screening was performed on 194 tetracycline-resistant colonies from four <i>E. coli</i> ESI123 strains carrying different pBACpAK constructs. The derivatives showed increased MGE capture rates (10.7-73.1 %) compared to the WT vector (3.75%), identifying multiple MGEs, including the novel composite transposon Tn<i>7824</i>. Tn<i>7824</i> harbours the <i>bla</i> _OXA-181 carbapenem resistance gene and the <i>qnrS1</i> quinolone resistance gene, highlighting the clinical relevance of these findings. Long-read sequencing of transposants confirmed the accuracy of MGE identification and structural characterization, which also revealed chromosomal integration events of the pBACpAK derivatives mediated by flanking insertion sequences. <b>Conclusions.</b> The modifications introduced in the pBACpAK derivatives could increase the detection of transposition events by alleviating spatial constraints, allowing for more robust MGE detection.